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Development of aptamer based HIV-1

entry inhibitor prophylactic drugs

Grace LondonCSIR, Biosciences

Emerging Health Technologies Platform

Pretoria, South AfricaPretoria, South Africa

2nd International Conference and Exhibition on Pathology

Properties of Aptamers

� Aptamers areantibodies

nucleic acids with properties of

� simple in vitro process called SELEXGenerated by

� High affinity and specificity� High affinity and specificity

� Small in size and fold in 3 –D structure (e.g RNAaptamers)

� Resistant to nucleases and chemically stableJoubert et al ., 2010

� Low toxicity and non(immunogenic

Applications of aptamers

Marro et al ., 2005 Rotherham et al ., 2012

Rusconi et al ., 2004

Green et al ., 2001

Appl. Microbiol. Biotechnol., 2005, Nov 11, 1-8

Anti-gp120 aptamers as HIV-1 entry inhibitors

Apt 2Apt 1

Apt 2 bind recombinant gp120Apt 1 bind gp120 trimer

Apt = aptamerKhati et al ., 2003; Cohen et al., 2008

HIV-1 entry and inhibitors

HIVHIV

HIV

gp120Co-receptor binding

gp41gp120- CD4 receptor binding

gp41gp120- CD4 receptorbinding

gp120

CD4

CCR5CXCR4 gp41 Mediated

Fusion

Host Cell

Pierson and Doms ., Current Top Microbiol Immunol (2003) 281 pp 1-27

Outline of the study

1. Evaluate efficacy of anti-gp120aptamer

subtype C

against

HIV-1

2. Testtoxicitytoxicity

3. Map “aptatope’s ”on

HIV-1gp120

4. Test

synergy with other entryinhibitors

AptamersC

inhibit entry of HIV-1 subtype

Env pseudoviruses

�

�

�

50 nM = No inhibitionNT = Not titred

* viruses using CXCR4 coreceptor

Aptamer IC50

Env clone Stages of disease Geographic location Apt 1 Apt 2

CAP45.2.00.G3 Acute/early S.Africa 2.6 0.3

ZM233M.PB6 Acute/early Zambia 4.9 0.1

ZM249M.PL1 Acute/early Zambia >50 0.6

ZM53M.PB12 Acute/early Zambia 20.1 0.8

ZM109F.PB4 Acute/early Zambia 14.1 >50

ZM197M.PB7 Acute/early Zambia 19.2 0.4

CAP210.200.E8 Acute/early S.Africa 2.2 0.1

ZM135M.PL10a Acute/early Zambia 2.8 NT

ZM214M.PL5 Acute/early Zambia 17.6 0.2

DU172.17 Acute/early S.Africa 12.1 0.6

DU156.12 Acute/early S.Africa 3.3 0.6

DU422.1 Acute/early S.Africa >50 0.3

CAP08.2.00.F6 Acute/early S.Africa 1.2 1

� Apt 1 = inhibited 84 % viruses� Mean IC50 6.6 ± 8.1 nM

� Apt 2 = inhibited 79 % viruses� Mean IC50 0.4 ± 0.3 nM

CAP08.2.00.F6 Acute/early S.Africa 1.2 1

CAP61.2.00.F10 Acute/early S.Africa 0.3 0.4

CAP63.2.00.A9J Acute/early S.Africa 1.7 0.1

CAP84.2.00.32J Acute/early S.Africa 29.1 >50

CAP85.2.00.09J Acute/early S.Africa 16.1 0.1

CAP239.2.00.G3J Acute/early S.Africa 7.1 0.2

RP1.12* Acute/early S.Africa 4.7 0.6

RP4.3 Acute/early S.Africa >50 0.5

COT6.15 Chronic S.Africa 0.9 >50

COT9.6 Chronic S.Africa 6.1 >50

DU151.2 Acute/early S.Africa >50 >50

DU123.6 Acute/early S.Africa >50 >50

Conc Acute/early S.Africa 5 0.1

CAP288.2.00.5 Acute/early S.Africa 9.5 0.3

CAP206.2.00.E8 Acute/early S.Africa 3.8 0.7

CAP244.2.00.D3 Acute/early S.Africa 2.3 0.4

RP6.6 Acute/early S.Africa 3.7 0.4

CAP88.2.00.B6J Acute/early S.Africa 1.6 1

IN8362.25 Acute/early India 18.2 NT

IN0013095.211 Acute/early India 0.2 NT

% viruses

neutralized 84% 79%

Mean IC50 (nM) 6.6 ± 8.1 0.4 ± 0.3

Aptamers inhibit entry ofPBMC

HIV-1 subtype C

Virus controlApt 2

Aptamer[nM] Aptamer][nM]

Mean IC80 in PMBC = 80 ± 11.8 nM

Aptamer[nM] Aptamer [nM]

UCLA1 (nM)

Virus Control

M

.5

.0

OT= 2

)lm

Aptamerssubtype

Du422IC80 = 6

nM

inhibit entry of HIV-1C in Macrophages

COT9IC80 = 29

nMDu422

COT97.5 2

UCLA1(nM)

VirusControl

UCLA1 (nM)

7.5

5.0

2

C 9

*** p <0.0019 nMVir*ups

<C0o.n0t5rolNS: notsignificant

*** p < 0.001* p <0.05NS: not

significant

NS IC801

5.02.5

Virus controlApt 21

2.50.0 *** p < 0.001

* p < 0.05NS: not significant

03.6 11 33� 100

Control

1 UCLA1 (nM)

3.6 11 33 100

Apt UCLA1 (nM)

CoNnStrol

Aptamer [nM]amer [nM]

0.0

2.5 03.6 11 3.6 11 33ADA100 Contr �

P24

(ng P

m 24l)

(ng

ml)

P24

(ng

ml)

p24

(ng

/ml) p24

(ng

/ml)

p24

(ng

/

IC80 = 29 nM

C

IC80

2�

IC80 = 6 nM

Du422IC80 = 6 n

7

5

NS

0 3.6 11

SW3314 100

IUCC8L0A=12(

n6Mn) M

3.6 11 33ADA100

Control

UCICL8A01=(n3M0) nM

0

Control

�

0.03 4033 100 ControlSW11

4ADA3.

6

3.6 11 33 100

UCLA1 (nM)

Control

Mean ICIC80 = 26UnCMLA1 (nM)

SW14

IC80 = 30

nMin MDM = 23 ± 10.4 nM30

8023 ADA40

IC80 = 30 nM20

1

230

10�

200 2

3.6 11 33 100 Control

3.6 11 33 100 ntrol1

��

�UCLA1(nM)

10�

�

0 � 0�

3.6 11 33 100 Contr�ol

3.6 11 33 100 Contro�l��

AptamU0CLA1 (nM)

���AptaUmCeLrA[n1M(n]M)

3.6 11 33 100

UCLA1 (nM)

er[nM]0

Control 3.6 11 33 100

UCLA1 (nM)

Control

p24

(ng/m

l) p24

(ng

/ml)

p24

(ng

/ml)

p24

(ng

/ml)

p24

(ng

/ml)

p24

(ng

/ml)

�4�0�

���

30 ���

0

IC80

Co

20

���

10 �����

IC80 = 26 nM

3

�

�

1

UCLA1 (nM)�

�

Aptamers exhibit no cytotoxicity

TZM-bl

MTS based-assayPBMC

MTS based-assay120 120

100 100

80 80

60 60

Ce

llV

iab

ility

Ce

llV

iab

ility

40 40

2020

00

%C

%C

Aptamers interact with conserved

residues on gp120

Aptamers bind to amino acids within the coreceptor (CoRbs) CCR5 binding site

Mufhandu, H et al., J. Virol. (2012,)86(9), pp. 4989

Synergy of aptamers withinhibitors

HIV-1 entry

100100UACpLtA21+Tb21

02UACpLtA21

TT2200

7575

T 20

5010010050

UCLA1+T20

UCLA1

T207525 25

5050 0 0

0.01 0.1 1 10 100 0.01 0.1 1 10 100

µM Lo2g5 Synergism : CI = 0.3 – 0.925

%in

hib

itio

n

ZM249

75

Du156 T 20

ZM249 T20Du156 T20

UCLA1+b12

UCLA1

b12

µM Lo2g5 Synergism : CI = 0.3 – 0.925

Antagonism : CI = >1Du172 b121

SW7 b120.1

00.01

100

0

0.010.1 10 100

100

1 10 100

UCLA1+b12

UCLA1

b1275 75

100 10050 Apt 2 + b12

Apt 2T20

50

75 7525 25

50 5000.01

00.1 1 10 100 0.01

2525

µM (Log)0 00.01 0.1 1 10 100 0.01 0.1 1 10 100

µM (Log)

%%

inIh n

ibh

i it %bio

in tIi n

ohn

i

bit

ion 0.1 1 10 100

µM (

SW7 b12Du172 b12

Summary

� Anti-gp120

RNA

aptamers

areefficacious

against

HIV-1subtype C isolates ( concentration in

nanomolar)

� They interact with conserved residues on gp120, delay virusresistance

� Not toxic in different cell types

� Synergy with other entry inhibitors, combination therapy withother drugs

� Anti-gp120 aptamers can be developed as entry inhibitordrugs

Acknowledgements

CollaboratorsFunding

•

•

•

•

NICD (Lynn Morris)

UKZN (Alexander Pym)

UCT & GSH (B. Mayosi)

University of Oxford, UK (WilliamJames)James)

• The Scripps Research Institute, USA (Dennis Burton)

Reagents

• Los Alamos National Lab, USA (Basil ISwanson)

•

•

IAVI

NIH AIDS Reagents

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